A selectable marker is a detectable genetic trait or segment of DNA that can be identified and tracked. A marker gene typically serves as a flag for another gene, sometimes called the target gene. A marker gene is typically used with a target gene being used to transform target cells. Target cells that heritably receive the target gene can be identified by selecting for cells that also express the marker gene. The marker gene is near enough to the target gene so that the two genes (the marker gene and the target gene) are genetically linked and are usually inherited together. The current standard for selectable markers is the “pat” gene which encodes an enzyme called phosphinothricin acetyl transferase.
Glutamine synthetase (“GS”) in many plants is an essential enzyme for the development and life of plant cells. GS converts glutamate into glutamine. GS is also involved in ammonia assimilation and nitrogen metabolism. GS is involved in a pathway in most plants for the detoxification of ammonia released by nitrate reduction. Therefore, potent inhibitors of GS are very toxic to plant cells. Breakdown or modification of the herbicide inside the plant could lead to resistance.
A particular class of herbicides has been developed, based on the toxic effect due to inhibition of GS in plants. Bialaphos and phosphinothricin are two such inhibitors of the action of GS and possess excellent herbicidal properties. These two herbicides are non-selective; they inhibit growth of all the different species of plants present on the soil, accordingly causing their total destruction.
Bialaphos is also a broad spectrum herbicide. Bialaphos is composed of phosphinothricin (PPT or PTC; 2-amino-4-methylphosphinobutyric acid), an analogue of L-glutamic acid, and two L-alanine residues. Thus the structural difference between PPT and Bialaphos resides in the absence of two alanine amino acids in the case of PPT. Upon removal of the L-alanine residues of Bialaphos by intracellular peptidases, the PPT is released. PPT is a potent inhibitor of GS. Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.
Bialaphos was first disclosed as having antibiotic properties, which enabled it to be used as a pesticide or a fungicide. U.S. Pat. No. 3,832,394 relates to cultivating Streptomyces hygroscopicus, and recovering Bialaphos from its culture media. However, other strains, such as Streptomyces viridochromogenes, also produce this compound. Other tripeptide antibiotics which contain a PPT moiety are also known to exist in nature, such as phosalacin. PPT is also obtained by chemical synthesis and is commercially distributed.
Bialaphos-producing Streptomyces hygroscopicus and Streptomyces viridochromogenes are protected from PPT toxicity by an enzyme with phosphinothricin acetyl transferase activity. Plant Physiol, April 2001, Vol. 125, pp. 1585-1590 (“Expression of bar in the Plastid Genome Confers Herbicide Resistance,” Lutz et al.). The Streptomyces species that produce these antibiotics would themselves be destroyed if they did not have a self-defense mechanism against these antibiotics. This self-defense mechanism has been found in several instances to comprise an enzyme capable of inhibiting the antibiotic effect.
Phosphinothricin acetyl transferase is encoded by either the bar (bialaphos resistance; Thompson et al., 1987) or pat (phosphinothricin acetyltransferase; Strauch et al., 1988) genes, and detoxifies PPT by acetylation of the free amino group of PPT. The enzymes encoded by these two genes are functionally identical and show 85% identity at the amino acid level (Wohlleben et al., 1988; Wehrmann et al., 1996). PPT-resistant crops have been obtained by expressing chimeric bar or pat genes in the cytoplasm from nuclear genes. Herbicide-resistant lines have been obtained by direct selection for PPT resistance in tobacco (Nicotiana tabacum cv Petit Havana), potato, Brassica napus, Brassica oleracea (De Block et al., 1987; De Block et al., 1989), maize (Spencer et al., 1990), and rice (Cao et al., 1992).
A gene (bar) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3. The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos producers Streptomyces viridochromogenes and Streptomyces hygroscopicus, respectively. The S. coelicolor bar gene conferred resistance to bialaphos when cloned in S. coelicolor on a high-copy-number vector. Bedford et al., Gene, 1991 Jul. 31; 104(1):39-45, “Characterization of a gene conferring bialaphos resistance in Streptomyces coelicolor A3(2).” Heterologous expression of this gene in other microbes, or transformation of this gene into plants, has not heretofore been reported.
The use of the herbicide resistance trait is referred to in DE 3642 829 A and U.S. Pat. No. 5,879,903 (as well as U.S. Pat. Nos. 5,637,489; 5,276,268; and 5,273,894) wherein the pat gene is isolated from Streptomyces viridochromogenes. WO 87/05629 and U.S. Pat. No. 5,648,477 (as well as U.S. Pat. Nos. 5,646,024 and 5,561,236) refer to the use of the bar gene from S. hygroscopiicus for protecting plant cells and plants from glutamine synthetase inhibitors (such as PPT) and to the development of herbicide resistance in the plants. The gene encoding resistance to the herbicide BASTA (Hoechst phosphinothricin) or Herbiace (Meiji Seika bialaphos) was introduced by Agrobacterium infection into tobacco (Nicotiana tabacum cv Petit Havan SRI), potato (Solanum tuberosum cv Benolima), and tomato (Lycopersicum esculentum) plants, and conferred herbicide resistance.